The present invention relates to cloning vectors and improved methods for inserting nucleic acid fragments into circular vectors. The invention further relates to improved methods of DNA library construction. The present vectors and methods allow minute amounts of nucleic acid fragments to be efficiently cloned. Moreover, the vectors and methods of the present invention avoid the size selection problems of currently available vectors and cloning methods. Thus, larger nucleic acid fragments are just as readily cloned using the methods and vectors of the present invention, as are smaller nucleic acid inserts. Accordingly, highly representative libraries can readily be made.
Circular vectors are popular and convenient vectors for isolating, maintaining and manipulating nucleic acid fragments. However, currently available methods of nucleic acid insertion into circular vectors have some serious disadvantages. Usually, the desired circular one vectorxe2x80x94one insert construct constitutes less than 0.1% of the products when current methods requiring DNA ligation, ligation-independent or topoisomerase joining reactions are used. The remaining 99.9% or more of the products formed include linear concatemers containing multiple vectors and/or multiple inserts. While this efficiency may be sufficient for simple subcloning experiments, it is unacceptable for libraries of complex populations of genomic DNA or cDNA.
One of the major problems of currently used methods is that reaction conditions which are optimized to encourage joining of an insert to a vector tend to discourage circularization of the vector-insert construct. Thus, if the concentrations of vector and insert are sufficiently high, the initial joining of one end of the vector with one end of the insert is a frequent event. However, circularization to form a vector with one insert is problematical because, at this high DNA concentration, the two free ends of the linear vector-insert construct are surrounded by many other DNA ends. Thus, the ends of the vector-insert construct are much more likely to be intermolecularly joined to other DNA ends than to each other. The major products formed are thus linear concatemers containing multiple vectors and/or multiple inserts. On the other hand, at the low DNA concentrations which would tend to facilitate circularization, the initial joining of the vector and insert becomes less likely. Many of the products formed under these conditions are therefore vectors without inserts. Hence, currently used methods are inefficient and can cause vector-to-vector ligation, low efficiency of nucleic acid insertion, and xe2x80x9cscramblingxe2x80x9d of different nucleic acid fragments, where two or more nucleic acid fragments are joined and inserted into the vector as though they were one fragment. These problems are particularly evident when the cloning reaction involves blunt-ended nucleic acids and complex mixtures of nucleic acids.
To obtain a reasonable number of the desired type of clones, currently used methods generally require optimization of the conditions used for insertion of a fragment into a vector. In practice, this means performing a series of pilot experiments using serial dilutions of each fragment population with each vector type, because optimal cloning conditions depend on the concentration and molar ratio of insert to vector, as well as the lengths of both the vector and fragment insert. No simple formula exists for optimizing the cloning conditions. And if the pilot experiments are not performed, conditions are generally far from optimal, providing only low numbers of clones and unrepresentative libraries.
Moreover, currently used methods strongly select for shorter fragment inserts. This occurs because the ends of longer vector-insert constructs are more likely to become joined to the ends of other vectors or inserts. In contrast, the ends of shorter vector-insert constructs are more likely to find each other and circularize than are the larger vector-insert constructs. The result is unrepresentative libraries which contain a higher proportion of smaller fragments than of larger fragments.
Accordingly, a need exists for new vectors and simplified methods that permit insertion and cloning of nucleic acid fragments and creation of representative DNA libraries.
The present invention provides a method for inserting a nucleic acid fragment into a circular vector, which includes:
(a) stably joining an insertion end of a nucleic acid fragment with an insertion end of a linearized vector at a first nucleic acid concentration under conditions favoring intermolecular joining, to form a linear vector-insert concatemer;
(b) melting hybridized cohesive circularization ends in said vector-insert concatemer to form a linear vector-insert monomer having single-stranded cohesive circularization ends; and
(c) reannealing said single-stranded cohesive circularization ends at a second nucleic acid concentration under conditions favoring circularization to form a circularized vector containing a nucleic acid insert;
wherein said second nucleic acid concentration is more dilute than said first nucleic acid concentration and wherein said cohesive circularization ends are between about 8 and about 50 nucleotides in length.
The present invention also provides a method for inserting a nucleic acid fragment into a circular vector, which includes:
(a) stably joining an insertion end of a nucleic acid fragment with an insertion end of a linearized vector at a first nucleic acid concentration under conditions favoring intermolecular joining, to form a linear vector-insert construct with complementary circularization ends, wherein one or both circularization ends of the vector-insert construct (1) are attached to an enzyme or enzyme complex capable of covalently joining DNA ends, and (2) are blocked from covalent joining;
(b) unblocking said circularization ends of the vector-insert construct; and
(c) joining the circularization ends of the insert-vector construct at a second nucleic acid concentration in an intramolecular reaction mediated by the enzyme or enzyme complex under conditions favoring circularization, to form a circularized vector containing a nucleic acid insert;
wherein the second nucleic acid concentration is more dilute than the first nucleic acid concentration.
The present invention is further directed to a nucleic acid insert in a circular vector which is prepared by the present methods. In a preferred embodiment, the present invention provides a genomic library or a cDNA library in a circular vector which is prepared by the present methods.
The present invention also provides a linearized vector which includes an origin of replication, an insertion site, and two complementary cohesive circularization ends, wherein:
each of said cohesive circularization ends is at least about 20 base pairs from said insertion site;
said cohesive circularization ends are between about 8 and about 50 nucleotides in length; and
upon hybridization ligase does not substantially covalently join said cohesive circularization ends.
The present invention further provides a linearized vector which includes an origin of replication, a blunt or short sticky insertion end, and a cohesive circularization end, wherein said short sticky insertion end is between 1 and 7 nucleotides in length and said cohesive circularization end is between about 8 and about 50 nucleotides in length.
The present invention also provides a vector including an origin of replication, an insertion end, and a cohesive circularization end, wherein:
said insertion end is covalently linked to a site-specific topoisomerase; and
said cohesive circularization end is between about 8 and about 50 nucleotides in length.
The present invention further provides a linearized vector which includes an origin of replication, two insertion ends, and two circularization ends wherein:
each of said circularization ends is located at least 15 base pairs from each of said insertion ends;
each of said insertion ends is covalently linked to a site-specific topoisomerase;
one or both of said circularization ends are covalently linked to a site-specific topoisomerase; and
each of said insertion ends and each of said circularization ends has a 5xe2x80x2-phosphate.
The present invention also provides a linearized vector which includes an origin of replication, a bacteriophage or virus cos site, and two insertion ends covalently linked to a site-specific topoisomerase.
The present invention also provides a kit which includes a first compartment containing the linearized vector of the present invention. The kit can also provide another compartment containing a DNA ligase, a terminase, a buffer including polyethylene glycol of high molecular weight, and/or a buffer which includes a salt.